Development of Sensitive and Specific Determination Methods of Bioactive Compounds Based on Generation of High-Performance Antibodies

Abstract

Immunoassays enable the sensitive determination of various compounds and have been widely utilized in pharmaceutical and medical sciences. To develop practical assays, it is essential to obtain antibodies that capture the target analytes with high specificity and affinity. To date, we have generated high-performance antibodies and developed immunoassays for determining bioactive compounds, particularly focusing on haptens, such as steroids and synthetic drugs. In previous studies, we have produced specific anti-hapten antibodies by immunizing animals with reasonably prepared hapten–carrier conjugates. However, the resulting antibodies sometimes lacked sufficient affinity for a sensitive determination. Therefore, we challenged genetic engineering to produce artificially modified antibodies with improved affinity. Therein, native antibodies with insufficient affinities were converted into single-chain Fv fragments (scFvs), to which random point mutations were introduced to generate diverse scFv libraries. Mutated scFv species with increased affinities were selected and isolated with the aid of phage-display system combined with panning. Using this strategy, we obtained scFvs specific to several haptens, such as estradiol-17β (E₂) and cotinine, that show significantly improved affinity (K_a) than that of the parental scFv, enabling more sensitive enzyme-linked immunosorbent assays. However, the panning step often fails in straightforward selection and requires laborious trial-and-error work. Thus, we developed a “clonal array profiling (CAP)” system for more efficient isolation of the mutants with enhanced affinities, which successfully functioned generating multiple anti-cortisol scFvs with the K_a improved up to 63-fold and an anti-E₂ scFv with 372-fold larger K_a. In this study, we identified new strategies that allow for efficient site-directed mutagenesis to improve affinity. We expect that the engineered antibodies described here will open the door to next-generation immunoassays that will enable simpler and more reliable determination of bioactive compounds.